Embryo Axis Research Articles

The Origin and Regulation of Neuromesodermal Progenitors (NMPs) in Embryos.

Neuromesodermal progenitors (NMPs), serving as the common origin of neural and paraxial mesodermal development in a large part of the trunk, have recently gained significant attention because of their critical importance in the understanding of embryonic organogenesis and the design of in vitro models of organogenesis. However, the nature of NMPs at many essential points remains only vaguely understood or even incorrectly assumed. Here, we discuss the nature of NMPs, focusing on their dynamic migratory behavior during embryogenesis and the mechanisms underlying their neural vs. mesodermal fate choice. The discussion points include the following: (1) How the sinus rhomboidals is organized; the tissue where the neural or mesodermal fate choice of NMPs occurs. (2) NMPs originating from the broad posterior epiblast are associated with Sox2 N1 enhancer activity. (3) Tbx6-dependent Sox2 repression occurs during NMP-derived paraxial mesoderm development. (4) The nephric mesenchyme, a component of the intermediate mesoderm, was newly identified as an NMP derivative. (5) The transition of embryonic tissue development from tissue-specific progenitors in the anterior part to that from NMPs occurs at the forelimb bud axial level. (6) The coexpression of Sox2 and Bra in NMPs is conditional and is not a hallmark of NMPs. (7) The ability of the NMP pool to sustain axial embryo growth depends on Wnt3a signaling in the NMP population. Current in vitro models of NMPs are also critically reviewed.

Overexpression of AtMYB12 transcription factor simultaneously enhances quercetin-dependent metabolites in radish callus

The study aimed to enhance quercetin production in radish by optimizing Agrobacterium tumefaciens-mediated in-planta transformation. This protocol involved infecting radish seed embryo axis with A. tumefaciens EHA105 strain carrying the 35S::AtMYB12. Radish seeds were infected with the Agrobacterium suspension (0.8 OD600) for 30 min, followed by sonication for 60 s and vacuum infiltration for 90 s at 100 mm Hg. A 3-day co-cultivation in Murashige and Skoog medium with 150 μM acetosyringone yielded a transformation efficiency of 59.6% and a transgenic callus induction rate of 32.3%. Transgenic plant and callus lines were confirmed by GUS histochemical assay, PCR, and qRT-PCR. The transgenic lines showed an increased expression of flavonoid pathway genes (AtMYB12, CHS, F3H, and FLS) and antioxidant genes (GPX, APX, CAT, and SOD) compared to WT plants. Overexpression of AtMYB12 in transgenic callus increased enzyme activity of phenylalanine ammonia lyase, catalase, and ascorbate peroxidase. In half-strength MS medium with 116.8 mM sucrose, the highest growth index (7.63) was achieved after 20 days. In AtMYB12 overexpressed callus lines, phenolic content (357.31 mg g−1 dry weight), flavonoid content (463 mg g−1 dry weight), and quercetin content (48.24 mg g−1 dry weight) increased significantly by 9.41-fold. Micro-wounding, sonication, and vacuum infiltration improved in-planta transformation in radishes. These high-quercetin-content transgenic callus lines hold promise as valuable sources of flavonoids.

Scanning Electron Microscopy Analysis of Tea’s Embryo Axis Explant Cultured on Murashige and Skoog Medium Containing 2,4-Dichlorophenoxyacetic acid

Camellia sinensis L. is an important crop in Indonesia as healthy beverage that contains several secondary metabolism compounds, such as polyphenols and catechins. Tissue culture including somatic embryogenesis and organogenesis has been used for propagating plant for various needs. In this present short-communication, scanning electron microscopic (SEM) analysis of tea was conducted and discussed. This study aimed to investigate surface ultrastructure of TRI2025 embryo axis tea clone cultured on Murashige and Skoog (MS) medium containing 2,4-Dichlorophenoxyacetic acid (2,4-D). The results revealed two different forms of explant’s development, i.e. somatic embryo and transitional form between somatic embryogenesis and organogenesis; or called by “Globular-like Structure” (GLS). Surface ultrastructure analysis of somatic embryo and GLS revealed respectively many stages of somatic embryo development i.e. globular, torpedo, and cotyledon stage, and leaf development form GLS regeneration.

Tissue and subcellular localization of CycD2 and KRPs are dissimilarly distributed by glucose and sucrose during early maize germination

In maize, immunoprecipitation assays have shown that CycD2;2 interacts with KRPs. However, evidence on CycD2;2 or KRPs localization and their possible interaction in specific tissues is lacking and its physiological consequence is still unknown. This work explores the spatiotemporal presence of CyclinD2s and KRPs, cell cycle regulators, during maize seed germination (18 and 36 h) after soaking on glucose or sucrose (120 mM). CyclinD2s are positive actors driving proliferation; KRPs are inhibitors of the main kinase controlling proliferation (a negative signal that slows down the cell cycle). Cell cycle proteins were analyzed by immunolocalization on longitudinal sections of maize embryo axis in seven different tissues or zones (with different proliferation or differentiation potential) and in the nucleus of their cells. Results showed a prevalence of these cell cycle proteins on embryo axes from dry seeds, particularly, their accumulation in nuclei of radicle cells. The absence of sugar caused the accumulation of these regulators in different proliferating zones. CyclinD2 abundance was reduced during germination in the presence of sucrose along the embryo axis, while there was an increase at 36 h on glucose. KRP proteins showed a slight increase at 18 h and a decrease at 36 h on both sugars. There was no correlation between cell cycle regulators/DNA co-localization on both sugars. Results suggest glucose induced a specific accumulation of each cell cycle regulator depending on the proliferation zone as well as nuclear localization which may reflect the differential morphogenetic program regarding the proliferation potential in each zone, while sucrose has a mild influence on both cell cycle proteins accumulation during germination. Whenever CycD2s were present in the nucleus, KRPs were absent after treatment with either sugar and at the two imbibition times analyzed, along the different embryo axe zones.

Elemental Mapping and Quantities in Different Soybean Seed Colors Using Micro X-Ray Fluorescence and Their Correlations with Germination

Micro X-ray fluorescence (μ-XRF) possesses a powerful analytical technique able to detect macro- and micro-elements. Each plant variety has a unique elemental composition and important role in the germination process. The aims of this study were to (1) map the elements and quantities in the soybean seed coat and endosperm, (2) investigate how the various elements might mediate the inter-relationship or correlation between elements within soybean seed genotypes with different seed coat colors, and (3) investigate that the targeted morphological characteristics especially in germination would be affected by seed elements. A μ-XRF technique was used for the elemental analysis and quantification. Three genotypes of Indonesian soybean were used in this study: greenish, black, and yellowish. In this study, we found that the silicon (Si) and magnesium (Mg) elements have a significant correlation. The high quantity of Si element in the embryo axis has a positive correlation with root length. The high quantity of Mg element which is evenly distributed on the endosperm has a positive correlation with normal germination. Si and Mg elements in the seeds have a negative correlation with imbibition water absorption. Based on the comparison between the three genotypes, the black genotype was superior in terms of germination and higher Si and Mg elements. Thus, the Si and Mg elements can be used as a reference in determining superiority of genotypes at the germination stage.

Genome-wide identification and expression analysis of the AUX/IAA gene family in turnip (Brassica rapa ssp. rapa)

BackgroundAuxin/indoleacetic acid (AUX/IAA) genes encoding short-lived proteins participate in AUX signaling transduction and play crucial roles in plant growth and development. Although the AUX/IAA gene family has been identified in many plants, a systematic analysis of AUX/IAA genes in Brassica rapa ssp. rapa has not yet been reported.ResultsWe performed a comprehensive genome-wide analysis and found 89 AUX/IAA genes in turnip based on the conserved AUX/IAA domain (pfam02309). Phylogenetic analysis of AUX/IAA genes from turnip, Arabidopsis, and cabbage revealed that these genes cluster into six subgroups (A1, A2, A3, A4, B1, and B2). The motif distribution was also conservative among the internal members of the clade. Enhanced yellow fluorescent protein (EYFP) signals of BrrIAA-EYFPs showed that BrrIAA members functioned as nucleoproteins. Moreover, transcriptional analysis revealed that the expression patterns of AUX/IAA genes in turnip were tissue-dependent. Because orthologs have similar biological functions and interaction networks in plant growth and development, BrrIAA66 in turnip possibly played a role in embryo axis formation, vascular development, lateral root formation, and floral organ development by interacting with BrrARF19 and BrrTIR1.ConclusionThese results provide a theoretical basis for further investigation of BrrAUX/IAA genes and lay the foundation for functional analysis of BrrIAA66 in turnip.

Spatially resolved transcriptomic analysis of the germinating barley grain.

Seeds are a vital source of calories for humans and a unique stage in the life cycle of flowering plants. During seed germination, the embryo undergoes major developmental transitions to become a seedling. Studying gene expression in individual seed cell types has been challenging due to the lack of spatial information or low throughput of existing methods. To overcome these limitations, a spatial transcriptomics workflow was developed for germinating barley grain. This approach enabled high-throughput analysis of spatial gene expression, revealing specific spatial expression patterns of various functional gene categories at a sub-tissue level. This study revealed over 14000 genes differentially regulated during the first 24 hafter imbibition. Individual genes, such as the aquaporin gene family, starch degradation, cell wall modification, transport processes, ribosomal proteins and transcription factors, were found to have specific spatial expression patterns over time. Using spatial autocorrelation algorithms, we identified auxin transport genes that had increasingly focused expression within subdomains of the embryo over time, suggesting their role in establishing the embryo axis. Overall, our study provides an unprecedented spatially resolved cellular map for barley germination and identifies specific functional genomics targets to better understand cellular restricted processes during germination. The data can be viewed at https://spatial.latrobe.edu.au/.

A Yap-dependent mechanoregulatory program sustains cell migration for embryo axis assembly

The assembly of the embryo’s primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development.

A protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil)

A reliable protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil) was developed. Using cultivar Laird, the protocol yielded rooted shoots from an average of 6.8 independent events per hundred seeds. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a β-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. A series of four media, each with appropriate levels of kanamycin selection were identified and other factors tested included the optical density of the Agrobacterium suspension, and type and concentration of plant growth regulators. The expression of the gus reporter gene was visualized through histochemical staining, and further molecular analysis through PCR, qPCR, ddPCR and Southern hybridization confirmed transformation and provided copy number. The inserted genes were inherited into the T1 generation and chimaeras were not identified. The time from co-cultivation to the planting of rooted shoots ranged from 4 to 7 months, as transgenic clusters continue to produce additional clonal shoots.

Revisiting the pericarp as a barrier restricting water entry/loss from cotyledons and embryonic axis of temperate desiccation-sensitive Quercus acorns.

Fully mature acorns of Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera are desiccation-sensitive. X-ray computer tomography showed that cotyledons shrink during drying, but embryos are protected. Information available on recalcitrant acorns of tropical and sub-tropical species of Quercus suggests that an impermeable pericarp, which limits the entry and loss of water only through the hilum (scar), is the underlying mechanism that prevents drying of the embryo axis following dispersal until the germination season. However, there is a lack of consensus supporting this proposition across species, and it is not well understood if such mechanisms occur in temperate Quercus species. This study investigated the significance of the acorn pericarp for temperate oak species and presents an ecological framework based on the post-dispersal climatic conditions. Using Quercus variabilis, Q. aliena, Q. mongolica, and Q. glandulifera acorns, the relationship between moisture content (MC) and germination was established, and X-ray computed tomography (X-ray CT) was used to understand the internal structural changes of cotyledons and embryonic axis occurring during desiccation. Water entry and exit routes through the scar, pericarp and apex were determined by imbibition and drying experiments. Climatic data and acorn morphological characteristics and germination were subjected to a principal component analysis (PCA). Freshly dispersed acorns of all species had a moisture content (MC) above 35% fresh weight (FW) basis, but drying to 15-10% MC resulted in complete loss of viability, implying recalcitrance behaviour. X-ray CT images suggested that the pericarp offers some protection to cotyledons and embryonic axis during desiccation, but it is contingent on MC. Extensive drying to a low MC with the scar and apex covered with vaseline resulted in internal tissues shrinkage, corresponding with viability loss. Water could enter or exit through the pericarp, albeit at a much slower rate than through the scar. A combination of factors including acorn anatomy, moisture content at the time of dispersal, microhabitat, the position of acorns in the soil prevent embryo desiccation below the critical MC and thus promotes survival of acorns on/in the soil during winter in temperate regions. Pericarp anatomy, to some extent, prevents excessive drying of the embryonic axis by slowing water movement, but prolonged drying or predatory pressure could result in pericarp cracks, favouring the absorption of water during sporadic rain. In the latter case, the survival of acorns possibly depends extensively on the continuous erratic rainfall, i.e. continuous wet-dry cycle, but in-situ experiments are yet to be performed to test this hypothesis.

Impact of environment on morphometric characteristics of pigeon pea (Cajanus cajan (L.) genotypes through seed digital imaging analysis

Computer seed digital imaging was utilized in this study to evaluate the differences in seed metric traits in twenty genotypes of pigeon pea as affected by the seed production environment. InSEEDLE™ software digital imaging analysis was used to differentiate the seed metric characters. For every replication, a hundred seeds were placed on the lighting hood in such a way that the embryo axis of the seed faced the image analysis system. Seeds were automatically analyzed by the scanner and the image of the seed was recorded by the 'WinSEEDLE™'. The procedure was repeated three times. The parameters observed were seed area, straight length, curve length, straight width, curve width, width length, and seed perimeter. Data collected were subjected to analysis of variance (anova) and principal component analysis. The result revealed that the pigeon pea genotypes evaluated were highly variable in all the seed metric traits evaluated. NSWCC-34C, NSWCC-34b, NSWCC-50 and NSWCC-34 were identified with consistent and high seed morphometric characteristic performance for most of the attributes examined, hence, an important tool in selecting superior pigeon pea genotypes and could be used as parental material.

In vitro callogenesis essays to induce somatic embryogenesis of the Tunisian chickpea

The present study was the first report on the somatic embryogenesis of the Tunisian chickpea (Cicer arietinum L.) particularly supposed to be recalcitrant and difficult to manipulate in vitro. An efficient protocol has been developed for inducing indirect somatic embryogenesis derived from immature zygotic embryo axis, young leaflet and hypocotylar explants. Callogenesis was achieved on full-strength Murashige and Skoog's (1962) (MS) basal medium supplemented with two auxin/cytokinin combinations; 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-furfurylamonopurine (KIN) or 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to establish the phytohormones requirement to promote the best induction of friable calli. For somatic embryos induction, embryogenic calli developed from zygotic embryo axes, leaflet and hypocotylar explants were cultured on MS full strength and MS/2, half strength, free of phytohormones media. This study found that all explants exhibited high frequency of callus induction. For immature zygotic embryo axes and young leaflet explants, media containing the combination of 2,4-D and KIN were best effective for inducing callogenesis and friable calli. The most important embryogenic callus percentages were obtained on the culture medium containing the highest concentrations of 2,4-D (2 mg L-1) and KIN (0.5 mg L-1). However, media containing the combination NAA and BAP were best effective for inducing embryogenic calli on hypocotylar explants. A maximal rate of embryogenic calli was reached on medium containing 3 mg L-1 NAA and 1 mg L-1 BAP. The highest number of somatic embryos was obtained with embryogenic calli derived from embryo axis explants and cultured on MS half strength free phytohormones medium. About 56% of somatic embryos converted successfully into fertile plantlets.

Microprobe-XRF Assessment of Nutrient Distribution in Soybean, Cowpea, and Kidney Bean Seeds: A Fabaceae Family Case Study

The present study explored microprobe X-ray fluorescence (XRF) spectroscopy for quantitative and space-resolved distribution of macro, i.e., potassium (K), phosphorus (P), sulfur (S), and calcium (Ca), and micronutrients, i.e., iron (Fe), zinc (Zn), and manganese (Mn) elemental composition in the cross-sectioned seeds of cowpea (Vigna unguiculata L.), kidney bean (Phaseolus vulgaris L.), and soybean (Glycine max L.) seeds, which are important agricultural species within the Fabaceae family. It unveils that both macro and micronutrients were heterogeneously distributed across seed tissues. Most of the P and S, Fe, Zn, and Mn were mainly found at the embryo axis tissues in all three Fabaceae species, whereas K was spread along the cotyledon and Ca was mostly observed trapped at the seed coat region. Furthermore, the Pearson correlation coefficient revealed a strong spatial correlation between P and S, and K and S in cowpea and soybean seed tissues, whereas Zn and Mn association was also recorded. Therefore, the μ-XRF technique is an important tool for assessing seed nutrient distribution, thus subsidizing the physiological role of nutrients in seeds and fostering innovative approaches for nutrient supply and biofortification.

Seed characteristics of Curculigo latifolia and its prospect to in vitro propagation

The propagation of Curculigo latifolia only occurs naturally in the wild and it is difficult to be domesticated via seed. It is suspected that the seeds are recalcitrant and require a special medium and care. In this study, we evaluated the structure of seed of C. latifolia using a stereo microscope, SEM and tetrazolium for seed viability test. The characteristics of the seeds observed in this study were the shape and color of the seeds, viability of the seeds with tetrazolim test. Seeds from the fresh fruit were cultured on MS0 medium to determine the ability of the seeds to germinate. This study showed that seeds aged 8-9 weeks after flowering were viable seeds with a red color indicated on all seed surfaces through the tetrazolium test.The structure of C. latifolia embryo is classified as an axial embryo with hypogeal germination type. Seeds of C. latifolia have potential to be used as explants for in vitro culture.

In vitro propagation of threatened therapeutic tree Buchanania lanzan Spreng (Anacardiaceae)

Embryo rescue refers to an in vitro technology have been used for propagating plants under in vitro from poorly developed embryos by avoiding embryo abortion. The aim of the present study was to in vitro regeneration of a medicinally wild tree species Buchanania lanzan through embryo rescue method. Isolated immature poorly developed embryo dissected out from de-coded seeds collected from 15 years old trees were cultured an Woody Plant Medium (WPM) was used to test in vitro germination. 100% embryo germination were recorded when the embryos inoculated on the medium supplemented with 1.5 mg/L-1 6-benzylaminopurine (BAP) and 50 mg/L-1 adenine sulphate (AS). Further, a week-old seedling derived explants viz embryo axis and cotyledonary node were selected to study in vitro clonal propagation. On medium augmented with 1.5 mg/L-1 6-benzylaminopurine (BAP) showed maximum mean shoots numbers (4.2±1.80) with 4.0±0.98 cm shoot height were achieved in cotyledonary node. Compare to Kinetin (Kn) BAP was to be better in shooting response and multiple shoot induction. Healthy regenerated shoots was separated from multiple shoots cluster and transferred to a rooting medium containing half strength WP medium fortified with Indole-3-butyric acid (IBA). IBA at 0.5 mg/L-1 showed 90% rooting response and maximum mean roots (4.9±0.98) per shootlet was obtained. The rooted in vitro plantlets were transferred to polythene bags containing autoclaved humus, red soil and farmyard manure (1:1:1 ratio) for acclimation at 25 ± 2 ᵒC. Later, these plantlets were gradually transferred to field condition. About 70% survival rate was recorded after three months period. The protocol developed in the present study was to restore the viable embryos from poorly developed immature embryos and rapid multiplication can help to ex situ conservation of this threatened tree B. lanzan.
 Keywords: Woody plant medium, In vitro seedlings, 6-benzylaminopurine, Adenine sulphate, Indole-3-butyric acid

Expression of radish defensin (RsAFP2) gene in chickpea (Cicer arietinum L.) confers resistance to Fusarium wilt disease.

Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity. In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms. Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea.

Effect of Environment on Morphometric Characteristics of Mung bean (vigna radiata (L.) Genotypes through Seed Digital Imaging Analysis

Digital imaging is a fast and reliable method for cultivar identification and discrimination. Computer seed digital imaging was utilized in this study to evaluate the differences in seed metric traits in ten genotypes of mung bean as affected by the seed production environment. A hundred seeds in each of the mung bean genotypes were subjected to digital imaging analysis using the ‘WinSEEDLE™’ software to differentiate the seed metric characters. For every replication, a hundred seeds were placed on the lighting hood in such a way that the embryo axis of the seed faces the image analysis system and the longitudinal axis runs parallel to the surface of the scanner. Seeds were automatically analyzed by the scanner and the image of the seed was recorded by the ‘WinSEEDLE™’. The procedure of hundred seeds placement on seed digital image was repeated three times for each genotype. The parameters observed were seed area, straight length, curve length, straight width, curve width, width length, and seed perimeter. Scan data collected from ‘WinSEEDLE™’ were subjected to analysis of variance and principal component analysis. The result revealed that the Mung bean genotypes evaluated were highly variable in all the seed metric traits evaluated. The study recommended that attention should be given to genotypes and seed production environments in the seed production of Mung bean. Genotypes Tvr-73, Tvr-27, Tvr-98, and Tvr-78 have been identified with consistent and high seed morphometric characteristic performance for most of the attributes examined, hence, can be important criteria in selecting superior seed physical traits and could be used as parental material, in the development of high seed yielding varieties.

DEVELOPMENTAL MORPHO-ANATOMY AND GERMINATION OF THE SEEDS OF Pterocarpus indicus f. echinatus Willd. VARIANTS

Previous studies on the embryo structure of legumes species had resulted in the division of the Fabaceae family into two great subfamilies based on embryo axis curvature. Research on seed morphology and anatomy adds to the knowledge of taxonomy, evolution and ecology. This study determined the seed developmental anatomy, pod and seed morphology as well as germination characteristics of the observed variants (T1 - small prickles; T2 - medium prickles; T3 - long prickles) of Pterocarpus indicus Willd. f. echinatus locally known as prickly narra in the Mount Makiling Forest Reserve (MMFR). Based on the anatomy of the root (radicle) and shoot apex, the formation of the leaf primordium in T2 seeds after radicle protrusion was more progressive. It was observed that the germination rate and the percentage were the highest in T2, where the apical dome was welldeveloped. The germination, pod and seed morphological characters as well as seed anatomical characters were proven to be systematically informative by showing significant differences among the variants.

Use of Emulsion-Templated, Highly Porous Polyelectrolytes for In Vitro Germination of Chickpea Embryos: a New Substrate for Soilless Cultivation

The application of highly porous and 3D interconnected microcellular polyelectrolyte polyHIPE (PE-PH) monoliths based on (3-acrylamidopropyl)-trimethylammonium chloride as soilless cultivation substrates for in vitro embryo culture is discussed. The embryo axes isolated from chickpea seeds are inoculated onto the surface of the monoliths and allowed to germinate. Germination study show that the newly disclosed PE-PH substrate performs much better than the conventionally used agar as the germination percentage, shoot and root length, fresh and dry weight as well as the number of leaves are enhanced. The PE-PHs exhibit a higher absorption capacity of the plant growth medium, that is, 36 g·g–1 compared to agar, that is, 20 g·g–1, and also survive autoclaving conditions without failing. The key advantage over standard agar substrates is that they can be reused several times and also without prior sterilization. These results suggest that PE-PHs with exceptional absorption/retention properties and robustness have great potential as soilless substrates for in vitro plant cultivation.

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS,1 being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC–MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60–260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60–140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.